Proposed research goals include (a) various aspects of inhibition of fatty acid synthetases by phenylmethylsulfonyl fluoride (PMSF); moles of C14-PMSF/mole of enzyme complex, sites of binding of palmityl group and sequence of palmityl- and PMS-peptides; (b) the study of the mechanism of initiation of fatty acid synthesis and release of products and the evaluation of nature of regions in the proximity of acyl binding sites and theioesterase catalytic site; (c) measurement of number of DPNH binding sites in the complex to distinguish between two types of TPNH binding sites and kinetic analysis of the order of addition of substrates and release of products; (d) interaction of antibodies to E. coli enzymes with the components of the complex and finally separation and characterization of individual enzymes. Tritium exchange, tryptophan fluorescence, circular dichroism, and differential centrifugation of native and PMSF modified enzymes in the absence and presence of acyl CoA compounds will be used to measure alteration of relative orientation of three covalent binding sites on binding acyl groups. Quenching of tryptophan fluorescence and equilibrium dialysis is to be used to measure DPNH binding to the enzymes. Binding of hydrophobic probes (naphthalene and acridine dyes) will be determined from difference spectrophotometry, fluorescence changes and equilibrium dialysis. Inhibition of binding of acyl groups and other model substrates in the presence of these hydrophobic probes wll be studied. Dissociation of mammalian fatty acid synthetase complex will utilize reagents (citraconic and 2,3-dimethyl maleic anhydrides) that react with NH3,-OH, and -SH groups and introduce net negative charges on the protein. Increased electrostatic repulsions due to large numbers of negatively charged groups has given indication of extensive dissociation of the complex. Separation of component enzymes will be attempted by affinity chromatography, isoelectric focussing and other methods.